Separation technology has evolved over centuries and is extensively applied in numerous industries, including such basic techniques as winnowing grain from chaff. Low cost separation processes are based on differences in gross physical properties, e.g., size, shape, density, oil/water solubility, and utilize a variety of physical forces or barriers such as gravity, centrifugation, flotation and sieving. More demanding separations are based on chemical and/or physicochemical properties which function at the molecular level, such as charge density and molecular size and shape. When entities are virtually identical at the gross and molecular level, affinity-binding is often relied on as the basis for separation. Affinity-binding discriminates at the molecular level, via a molecular "lock & key" mechanism, which is commonly referred to as a specific binding pair, e.g. ligand/receptor, interaction. The immune system in which antibodies and/or cells attack and destroy specific pathogens, as well as the endocrine system, in which hormones secreted by one gland create physiological effects in target tissue elsewhere in the body, are examples of nature's use of the affinity-binding principle.
Affinity-binding technology often makes use of receptors such as polyclonal antibodies and lectins, as well as hapten-labeling of molecules for recognition by anti-haptens. The emergence of monoclonal antibody technology has made affinity-binding separations a cost-effective and efficient reality. The next generation of affinity-binding technology will likely include the use of single chain antibodies, peptides and oligonucleotides or a combination thereof. Single chain antibodies (scFv) are engineered proteins which may be expressed on the surface of phages such as M13 or fd, and which bind to antigenic determinants in a manner similar to monoclonal antibodies which are traditionally generated from hybridomas. Relatively short peptides can also contain a binding site capable of discriminating antigenic determinants and can be further linked to small immuno-specifically recognizable substances, which effectively replace the "conserved" region of an antibody, and thus can themselves be immuno-specifically bound using a "second antibody" capture technique. Oligonucleotides may be chemically conjugated to either peptides or small molecules, and the resulting conjugate can specifically bind to antigenic determinants. In other cases, the oligonucleotide alone can bind to an antigenic determinant with high specificity. In order to obtain the desired specificity of these antigen binding probes, libraries have been constructed which contain a large diversity of such probes. Antigen binding probes can be isolated from such libraries by presentation of the appropriate antigen. After isolation of the antigen binding probes with the desired specificity, the probe can be further characterized and large quantities can be produced. See, for example, De Kruif, et al., PNAS in press; De Kruif, et al., JMB, in press; Chen & Gold, Biochemistry, 33(29):8746-56 (1994); Fodor, et al., Science, 251:767-73 (1991); and McCafferty, Nature, 348:552-54 (1990).
Separations using affinity-binding techniques are well established. There are numerous procedures available for separating biological entities such as cells, cell organelles and other cell components, viruses, bacteria, proteins and polynucleotides. Many of these procedures are dependent on transient binding of a target substance to a receptor on a solid support. Affinity-binding separation is commonly used in the purification of antibodies. Many commercially available affinity chromatography systems use polymeric beads linked to protein A or G, which specifically bind antibody, releasing the antibody upon flushing the column with low pH buffers. Other affinity-binding systems capture biological molecules, only to release the captured substance upon the introduction of another substance which can displace the entity from the support via competitive binding. Ionic strength manipulation may also be used to remove a specifically bound substance from an affinity separation device, such as a column.
Historically, binding agents having specific binding affinity for various analytes, e.g., antigens or antibodies, were attached to a solid support such as a beaker or a micro-titre plate, and placed in contact with the test sample, after which the unbound sample components were physically removed, thereby capturing the analyte of interest from the test medium. Other stationary supports include test tubes, membranes, gels and filter media. Mobile solid supports, such as latex beads, or beads made from other polymeric material are widely used to provide increased surface area on which to anchor a specific binding substance. Magnetic particles are advantageously used to facilitate separations. Magnetic particles ranging in size from 0.01-6 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; 4,659,678 and 4,795,698. Relatively large magnetic particles, such as the 4.5 micron sized particles marketed by Dynal (Oslo, Norway) respond to weak magnetic fields and magnetic gradients and are used for many types of biological separations. Because of their size, such particles tend to settle rapidly from solution and also have limited surface area per unit weight. Larger magnetic particles also tend to aggregate after they have been subjected to a magnetic field for a variety of reasons. Smaller sized magnetic particles, on the order of 0.01-0.8 microns, generally fall into two broad categories. The first category includes particles that are permanently magnetized and the second includes particles that are magnetically responsive only when subjected to a magnetic field. The latter are sometimes referred to as superparamagnetic particles. However, certain ferromagnetic materials, e.g., magnetic iron oxide, may be characterized as superparamagnetic when the crystal size is about 30 nm or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnetic characteristics after exposure to a magnetic field and tend to aggregate thereafter. See P. Robinson, et al., Biotech Bioeng, XV:603-06 (1973).
Various methods have been proposed to effect the release of cells or other target substances from a solid support once they have been separated. Giaever (U.S. Pat. No. 3,970,518) discloses the use of antibody-coated magnetic microspheres to separate cells, but uses a chemical cleaving agent such as formic or sulfuric acids to release the separated cells. Hayman, et al. (U.S. Pat. No. 4,988,621) discloses the use of a short peptide which interferes with the binding of cells to fibronectin, allowing the detachment of cells from a solid support. Mori (EP 463508 A) discloses the use of a temperature-responsive adhesive to immobilize a cell for microinjection, lowering the temperature to release the immobilized cell. Berenson (WO 91/16452) describes a technique involving agitation of an avidin column to remove cells captured thereon. The use of ionic strength manipulation to reversibly immobilize antibodies bound to magnetic beads has also been reported. See, for example, Scouten, Anal. Biochem. 205:313-18 (1992). Civin (U.S. Pat. No. 5,081,030) discloses the use of chymopapain to digest the cell surface antigen My 10, releasing stem cells from magnetic particles used to isolate the stem cells from a cell suspension. Berge et al. (WO 94/20858) describes the separation of target substances by means of a relatively large magnetic particle linked via a hydroxyboryl/cis-diol bond to an antibody, which bond is cleaved after separation of the target substance. The commercially available DETACHaBEAD from Dynal (Oslo, Norway) comprises an anti-mouse FAb with higher affinity for the binding site of a monoclonal antibody than the monoclonal has for its corresponding antigen. Therefore, the anti-FAb antibody can displace the original MAb from a target cell. See Geretti, et al. J. Immunol. Meth. 161:129-31 (1993); and Rasmussen, et al., J. Immunol. Meth. 146:195-202 (1992). Kessler (WO 94/02016) describes the use of an excess of soluble hapten to disrupt a hapten-antihapten complex, thereby releasing a cell from its solid support. See also Clark et al., J. Immunol. Meth., 51:167-70 (1982).
All of the above are generally satisfactory methods for the separation of a target substance from non-target substances, but the methods lack the capability to separate a selected subset of biological entities, or several selected subsets in sequence, from a bound mixed population thereof. Currently available biological separation technology enables only the separation or removal of a single target substance, leaving various non-target substances behind (positive selection), or the removal of various non-target substances, leaving a single target substance behind (negative selection). Such existing separation techniques are thus limited to the separation of a mixed population of biological entities into essentially two groups, target and non-target. There are certain drawbacks inherent in using a series of positive selection separations to separate more than one class of target substance from a mixture containing several non-target substances, based on existing separation techniques. Each interaction of biological entity with a specific receptor requires a finite time to reach equilibrium. Moreover, each step involved in such a separation is accompanied with variable degrees of loss of target entities and capture of non-target entities, no matter what type of specific procedure is employed. The time demands, and associated expense of multiple incubations, separations and restoration of the separation apparatus for each subsequent selection are also not insignificant. A technically feasible, economic technique for isolating one or more selected target substances from a mixed population thereof remains a highly desired goal which has not yet been satisfactorily realized.
Multi-parameter separations have been achieved in the last decade with the advent of fluorescent activated cell sorting. Cells are passed individually through a measurement orifice by means of hydrodynamic focusing. A light beam (such as a laser) is focused on the cell stream and the light absorbed, scattered or emitted is measured from each passing cell event. Various parameters can be measured of each cell passed through the orifice, including forward light scatter (a measure of cell size), orthogonal light scatter (a measure of cell granularity), depolarized light scattering (a measure of large intra cellular granules) and fluorescence. The fluorescent signals which can be potentially measured depend on the fluorescent probe, the wavelength(s) of the laser(s) and the spectral separation of the fluorescent probes used. Monoclonal antibodies specific for different antigens can be labeled with fluorochromes which can be spectrally separated and cells exhibiting different combinations of these cell surface antigens can thus be discriminated from each other. After identification of the cells, they can be separated by either mechanical separation or more conventionally by deflection of charged droplets which contain the cells with desired specificity. Although flow cytometry is an extremely powerful tool to identify and select cell sub-populations from a mixture of cells with a high degree of certainty, flow cytometers are limited by software and hardware requirements related to the passage of single cells. The highest reported cell rate which can be processed via flow cytometry is 40,000 cells/second, although commercially available instruments typically function at a speed which is ten-fold lower. See, Shapiro, Practical Flow Cytometry, Chapters 3 and 6 (1995). Additionally, cell loss can be significant, and as cell purity increases, the cell loss further increases. Furthermore, only two populations can be separated by a flow cytometer at a time, so that sorting cells into more than two populations requires multiple runs through the cytometer further exacerbating the cell loss. The complexity of these instruments also makes them expensive to acquire and maintain. The only practical option available to those interested in using flow cytometry appears to be the removal of the bulk of cells, then using flow cytometers to analyze the remaining portion of cells. This is the approach taken, for example, by Bolton, et al (WO 94/25852) with the removal of 95% of the leukocytes from a solid tumor sample with CD45 antibody. Although this approach overcomes the above-noted limitations of flow cytometers with respect to throughput, a more economical procedure enabling the separation of cells en masse is still a desired objective for various applications in scientific research, environmental analysis, food testing, forensic science, but most importantly in the medical field for the separation of different types of cells for diagnostic and therapeutic purposes, including gene therapy.